There is a high demand to determine the concentration of analytes of interest, which can be of environmental concern, such as fungal or microbial toxins (mycotoxins), which are fungal metabolites present in a large part of the world's food supply and pose a potential threat to food safety. In addition to this there is also a high demand to determine the concentration of analytes of interest in a sample of a mammal, such as drugs, steroids, hormones, proteins, peptides, lipids, sugars, receptors, nucleic acids, vitamins, etc, to identify an intoxication, control the medication of therapeutic drugs with a narrow therapeutic window, etc.
Assays, and in particular immunoassays, have been used in an effort to improve upon the success in detecting analyte substances at very low levels. For example, the use of such techniques has been prompted by the extraordinary successes that have been achieved in the measurement of biological substances by specific (immunological) binding moieties and techniques. Available evidence indicates that specific binding moieties, in particular antibodies, can be obtained even against low molecular weight organic compounds, such as pesticides or other haptens.
Any means of applying an (immuno-)chemical reaction to a detection problem ultimately relies upon a binding reaction occurring between analytes of interest and its specific binding moiety. One means by which this interaction can be employed in measurement and detection has come to be known as “competitive binding assay”. In principle, this method requires two reagents in addition to the sample to be tested. These two reagents are a labeled form of the analyte to be detected or measured in a fixed concentration, and a binding moiety, preferably antibody or receptor, specifically directed against the analyte. The principle of this assay involves a preliminary measurement at one given time point of the binding of the labeled analyte (substance being detected) with its binding moiety and then, a determination at the given time point of the extent of the inhibition of this binding by known quantities of the unlabeled analytes of interest in the sample, which corresponds to the unknown concentration of the analytes of interest. From these data, a standard curve at the given time point can be constructed which shows the degree of binding of the labeled analyte under certain specified conditions as a function of concentration of the unlabeled analytes of interest added.
Another means by which this interaction can be employed in measurement and detection has come to be known as “non-competitive binding assay”. In principle, this method requires only one reagent in addition to the sample to be tested. This reagent is a labeled form of the binding moiety, preferably antibody, receptor or enzyme, specifically directed against the analytes of interest. In principle this assay involves a direct measurement at one given time point of the binding of the labeled binding moiety with the analytes of interest in the sample followed by a determination at a given time point of the extent of intensity. From this data, a standard curve at the given time point can be constructed which shows the degree of binding by the labeled binding moiety with the analytes of interest under certain specified conditions as a function of concentration of the analytes of interest.
One way of implementing such (immuno-)assays is to employ a fluorescent label. Usually, fluorescent labeling of one of the reagents, e.g. the analyte used in known concentration, is important in carrying out the assay by means of fluorescence polarization and/or fluorescence intensity measurements. Unlike other assays, such as ELISA, no physical separation of bound form of the labeled analyte from free form is necessary. Therefore, a simple rapid optical measurement yields the essential information without physical separation of bound and free labeled materials.
Direct readout polarometer (having a machine time-constant of 0.1 seconds to several minutes) can be used to study slow kinetic reactions (reaction time-constant 10 seconds or longer) as well as reactions near or at equilibrium. These direct readout polarometer (defined as “static” polarometer) are capable of measuring both the degree of fluorescence polarization, P=(V−H)/(V+H) and the sum of intensities of polarized fluorescence in horizontal and vertical direction (V+H). V−H (the absolute difference of the intensities of polarized fluorescence in horizontal and vertical direction) can also be measured and utilized as a parameter. Some binding moiety—analyte reactions can be slow enough such that they can be studied with the static polarometer. Other binding moiety-analyte reactions occur too rapidly (reaction milliseconds to seconds) to be monitored by the static fluorescence polarization or fluorescence intensity device. Fast reaction technology (e.g. stopped-flow methodology) has been combined with fluorescence polarization and fluorescence intensity techniques to study rapid binding moiety-analyte, preferably hapten-antibody, rapid antigen-antibody, rapid enzyme-substrate, rapid substrate-receptor reactions. Such rate assays should lead in principle to simplified and improved assays even when applied to the analysis of real analytes. Yet currently there are few fluorescence polarization or fluorescence intensity rate immunoassays as well as other rate assays involving substrates and receptors. This is because fluorescence polarization and fluorescence intensity stopped-flow devices are expensive, somewhat complicated, and at times limited by background problems. “Static” fluorescence polarometer rate immunoassays require large dilutions of fluorescent reactants and analytes to slow down these fast reactions so that a reasonable time frame (seconds to minutes) can be attained.
As mentioned already above, in conventional competitive or non-competitive fluorescence labeled homogenous assays the concentration of the analytes is determined at one predetermined time point of the reaction. Normally, the time point of establishing the concentration of the analytes of interest in the sample correlates to the equilibrium condition of the reaction or another predetermined time point t=t0+x (with t0=the time point of addition of the last reaction component to the mixture and x=a time point of reaction) close to equilibrium condition. Thus, the time for measuring immuno-assays may take 5 minutes or more according to conventional procedures.
The sensitivity of assays, in the meaning of dependency of the concentration to the measured signal, has usually one optimal time period for a first concentration and a second optimal time period for a second concentration, where the margin of error is within a tolerable range. Due to the fact that only one measurement is taken at one time point, measuring errors and intolerable margin of error have a direct (intolerable) impact on the determined concentration of analytes in the sample.
In particular, when the determination of the concentration of analytes of interest is of environmental or health concern, there may be an interest that the tolerable margin of error is comparatively small and that the concentration is reliably determined.
Thus, there is a need to provide quick and reliable methods for determining the concentration of analytes of interests in a sample, wherein the margin of error is reliably within a tolerable limit. This will allow a person skilled in the art to provide a quick and reliable determination of concentration of analytes of interest, which may be in particular of environmental or health concern, so that reasonable precautions or counteractions may be undertaken.